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First published online 4 November 2008
doi: 10.1242/jcs.040477

Journal of Cell Science 121, 3867-3877 (2008)
Published by The Company of Biologists 2008
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Sarcomeric actin organization is synergistically promoted by tropomodulin, ADF/cofilin, AIP1 and profilin in C. elegans

Sawako Yamashiro1,*, Elisabeth A. Cox2,{ddagger}, David L. Baillie3, Jeff D. Hardin2 and Shoichiro Ono1,§

1 Department of Pathology and Department of Cell Biology, Emory University, Atlanta, GA 30322, USA
2 Department of Zoology, University of Wisconsin, Madison, WI 53706, USA
3 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6


Figure 1
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  		Fig. 1. Mutation of unc-94/tmd-1 causes severe disorganization of actin filaments in body-wall muscle. (A) Genomic organization of the unc-94/tmd-1 gene. Exons are indicated by boxes. Two alternatively spliced isoforms are generated as described (Stevenson et al., 2007Go). The position of the unc-94/tmd-1(tm724) deletion is indicated. (B) Western blot analysis of the TMD-1 protein in wild-type and unc-94/tmd-1(tm724) adult worms. The bands at ~68 kDa (arrow) are not related to the TMD-1 protein (Stevenson et al., 2007Go). Anti-actin antibody was used to monitor equal loading of the protein samples. (C) Actin filaments in embryos (a,b) and adults (c,d) were visualized by staining with tetramethylrhodamine-phalloidin in wild-type (a,c) and unc-94/tmd-1(tm724) (b,d). Bars, 10 µm. (D) Organization of {alpha}-actinin (a-f) and myosin (g-l) in body-wall muscle. Wild-type (a-c and g-i) or unc-94/tmd-1(tm724) (d-f and j-l) were immunostained for actin (a,d,g,j) and {alpha}-actinin (b,e) or MyoA myosin heavy chain (h,k). Merged images are shown in c, f, i and l (actin in red and {alpha}-actinin or MyoA in green). Arrows indicate positions of actin aggregates where {alpha}-actinin or myosin did not localize. Bar, 10 µm.

 

Figure 2
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  		Fig. 2. TMD-1 inhibits UNC-60B (ADF/cofilin)-dependent depolymerization of actin filaments in vitro. (A,B) Depolymerization of CapZ-capped actin filaments in the presence of Lat-A was examined by a pelleting assay. CapZ-capped F-Ce-actin (5 µM actin, CapZ:actin ratio of 1:100) was incubated with various combinations of TMD-1, CeTM, and UNC-60B in the presence of 10 µM Lat-A for 15 minutes. The mixtures were ultracentrifuged and fractionated into supernatants (sup) and pellets (ppt) and analyzed by SDS-PAGE (A) and densitometric quantification of depolymerized actin (%) in the supernatants (B). Data are the mean ± s.d. of three experiments. (C) Kinetic measurements of depolymerization of CapZ-capped actin filaments. CapZ-capped F-Ce-actin (CapZ: actin ratio of 1:100; 10% labeled with pyrene) was diluted to 0.5 µM actin with 1 µM Lat-A in the presence of indicated combinations of UNC-60B, CeTM, and TMD-1. Depolymerization of actin was monitored by decrease in pyrene fluorescence.

 

Figure 3
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  		Fig. 3. unc-94/tmd-1(RNAi) strongly enhances actin disorganization in unc-60B mutant backgrounds. (A) Effect of unc-94/tmd-1(RNAi) on the TMD-1 protein level. Wild-type worms were treated with control dsRNA or unc-94/tmd-1(RNAi) and the TMD-1 protein level was determined by western blotting using anti-TMD-1 antibody. Anti-actin antibody was used to monitor equal loading of the protein samples. (B) Localization of TMD-1 and UNC-60B in unc-60B mutant and unc-94/tmd-1(RNAi) muscles, respectively. TMD-1 (a,d) and actin (b,e) were immunolocalized in the body-wall muscle of wild-type (a-c) and unc-60B(r398) (d-f). Merged images are shown in c and f (TMD-1, green; actin, red). UNC-60B (g,j) and actin (h,k) were immunolocalized in the body-wall muscle of wild-type (g-i) and unc-94/tmd-1(RNAi) (j-l). Merged images are shown in i and l (UNC-60B in green and actin in red). (C) Effects of unc-94/tmd-1(RNAi) on worm motility in unc-60B backgrounds. Wild-type and four unc-60B alleles were examined. m35 and s1309 are strong loss-of-function, whereas r398 and s1307 are weak loss-of-functinon. Data are the mean ± s.d., n=10. Asterisks indicate P<0.005 by t-test comparing control RNAi experiments and unc-94/tmd-1(RNAi) for each strain. (D) Appearance of wild-type (a, f) and unc-60B (b-e, g-j) worms treated with control dsRNA (a-e) or unc-94/tmd-1(RNAi) (f-j) on agar plates. (E) Actin filament organization in the body-wall muscle of wild-type (a and b) and unc-60B (c-j) treated with control dsRNA (a,c,e,g and i) or unc-94/tmd-1(RNAi) (b,d,f,h and j). Arrowheads in b indicate actin aggregates. Arrows in c, e, g and i indicate striated organization of actin filaments.

 

Figure 4
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  		Fig. 4. unc-94/tmd-1(RNAi) strongly enhances actin disorganization in an unc-78-null background. (A) Localization of TMD-1 and UNC-78 in unc-78 mutant and unc-94/tmd-1(RNAi) muscles, respectively. TMD-1 (a,d) and actin (b,e) were immunolocalized in the body-wall muscle of wild-type (a-c) and unc-78(gk27) (d-f). Merged images are shown in c and f (TMD-1, green; actin, red). UNC-78 (g,j) and actin (h,k) were immunolocalized in the body-wall muscle of wild-type (g-i) and unc-94/tmd-1(RNAi) (j-l). Merged images are shown in i and l (UNC-78 in green and actin in red). (B) Effects of unc-94/tmd-1(RNAi) on worm motility in an unc-78 null background. Wild-type and unc-78(gk27) were examined. Data are the mean ± s.d., n=10. Asterisks indicate P<0.005 by t-test comparing control RNAi experiments and unc-94/tmd-1(RNAi) for each strain. (C) Appearance of wild-type (a,c) and unc-78(gk27) (b,d) worms treated with control dsRNA (a,b) or unc-94/tmd-1(RNAi) (c,d) on agar plates. (D) Actin filament organization in the body-wall muscle of wild-type (a,b) and unc-78 (c,d) treated with control dsRNA (a,c) or unc-94/tmd-1(RNAi) (b,d).

 

Figure 5
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  		Fig. 5. unc-94/tmd-1(RNAi) enhances the formation of actin aggregates in profilin-mutant backgrounds. (A) Frequency of adult worms that have actin aggregates in the body-wall muscle. Adult worms were stained with tetramethyl-Rhodamine–phalloidin, and worms with actin aggregates in their muscle were scored. Wild-type, pfn-2(null) and pfn-3(null) single mutants, and a pfn-3 pfn-2 double mutant were treated with control dsRNA or unc-94/tmd-1(RNAi). (B) Actin filament organization in the body-wall muscle of wild-type (a,b), pfn-2(null) (c,d), pfn-3(null) (e,f) and pfn-3(null) pfn-2(null) (g,h) treated with control dsRNA (a,c,e,g) or unc-94/tmd-1(RNAi) (b,d,f,h).

 

Figure 6
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  		Fig. 6. Mutation of profilin does not enhance the unc-60B phenotype. Actin filament organization in the body-wall muscle of wild-type (+;+) (A,B), +; pfn-3(null) pfn-2(null) (C,D), unc-60B(r398); +(E,F), unc-60B(r398); pfn-3(null) pfn-2(null) (G,H), unc-60B(s1309); + (I,J) and unc-60B(s1309); pfn-3(null) pfn-2(null) (K,L) treated with control dsRNA (A,C,E,G,I,K) or unc-94/tmd-1(RNAi) (B,D,F,H,J,L).

 

Figure 7
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  		Fig. 7. Model of synergistic regulation of sarcomeric actin organization by Tmod, ADF/cofilin, AIP1 and profilin. A stable thin filament has CapZ at the barbed end, Tmod at the pointed end, and tropomyosin on the side (A). Dynamic behavior of Tmod at the pointed end transiently generates a free pointed end (B) and allows elongation (C). This tropomyosin-free region of the filament is subjected to severing by ADF/cofilin and AIP1 (D), and depolymerized actin is captured by profilin, which prevents elongation from the pointed end (E). When this machinery is impaired, excessively elongated filaments may become unstable and form aggregates (F).

 

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© The Company of Biologists Ltd 2008